Name: hsv-mediated transgene expression of chimeric constructs to study behavioral function of gpcr heteromers in mice
Uploaded: 2016-07-09T13:00:00.000+00:00
Duration: 7 min 30 s
Description: Watch this Scientific Journal Video about HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice at JoVE.com

NIH R01MH084894参与这项研究的经费。我们想感谢博士。优思明赫德和斯科特·鲁索在西奈山医学院学校小鼠的捐赠，这项工作的拍摄过程中使用其手术和行为的设施。

注:动物养殖和关心所有程序均按照西奈山伊坎医学院的机构动物护理和使用委员会（IACUC）的规定进行。一定要在整个过程中使用无菌手套。 1.药物和病毒制剂<ol><li> 药物研制 <ol><li>通过溶解1.35毫升100mg / ml的氯胺酮和0.75毫升20毫克/毫升甲苯噻嗪12.9毫升0.9％盐水溶液制备15.0毫升氯胺酮/赛拉嗪麻醉剂。彻底混合的解决方案。 </li></ol></li><li> 病毒制剂 <ol><li>克隆mGlu2和mGlu2ΔTM4N构造成双顺反子单纯疱疹病毒（HSV）载体以下先前描述6标准协议。如前所述6,13,14包装病毒颗粒。残留的替代丙氨酸- 677 4.40，丙氨酸- 681 4.44和4.48 Ala685在mGlu2为Ser686 4.40，pH值E690 4.44和甘氨酸-694在4.48 mGlu3（HA-mGlu2ΔTM4N）先前已经6中所述。 注意:这是以前表明，嵌合构建的HA-mGlu2ΔTM4N在含有完整G蛋白依赖的信号6质膜表达。 </li><li>在不使用时在商店病毒载体-80℃。在冰上解冻病毒载体，然后分装到10微升等分试样。对于手术过程中，保持在冰上。 </li></ol></li></ol> 2.手术<ol><li> 术前准备 <ol><li>权衡鼠标和氯胺酮/甲苯​​噻嗪鸡尾酒的适宜剂量注入小鼠（详见1.1.1）。 </li><li>检查小鼠，看是否正常麻醉，挤压脚和尾对疼痛的反应，如果无法引起反应，鼠标被适当麻醉。 </li><li>从头骨的基地使用快船鼻尖剃光头鼠标。应用眼用凝胶鼠标AFterward防止小鼠的失明。 </li><li>加载每个注射器上的立体框架。然后倾斜立体框架的每个臂的垂直部分，从而它们是从正常的10度距离。确保臂倾斜，使得针彼此面对。 </li><li>填充用70％乙醇将针清洁每个注射器。填充针至少三次，以确保该注射器是干净的。 </li><li>一旦针头已被清洗，通过用双蒸H 2 O填充针冲洗针一旦刷新，填写每针1.3微升双蒸H 2 O的拧在注射器的柱塞释放0.3微升双蒸H 2 O的如果水珠在针尖，小心翼翼地擦去水。如果没有散发出来的注射器，推动柱塞完全放下，然后重复注射器清洗。 </li><li>充水后，再拉起注射器填充注射器0.5微升空气。 </li><li>一旦空气和水的注射器，认真填写注射器用1.3微升病毒解决方案。在这点上保证在注射器中的总体积为2.8微升。再次扭动注射器的尖端释放0.3微升病毒。如果在针尖液珠，仔细拭去液体。如果没有散发出来的注射器，推动柱塞完全放下，然后重复注射器清洗。 </li></ol></li><li> 手术 <ol><li>装上鼠标的立体框架，确保使头骨是水平的平调整立体框架。适用povodine碘到暴露的头皮。使用手术刀，使沿着露出剃区域内的颅骨的中线矢状切口。然后装上夹子滴定管对皮肤的切口部位，以确保头骨保持暴露。 </li><li>使用H 2 O 2溶解掉骨膜暴露的缝线头骨。现在，前囟门和缝合线是可见的，一定要调整立体框架，以确保头骨是水平的。 </li><li>对准前囟的注射器的针的提示和记录前囟的坐标。计算的，其中针将要插入的坐标。 <ol><li>对于延髓-Cauldal（RC）面，加1.6毫米将录制的前囟门RC坐标（+1.6从囟门）。对于背腹（DV）飞机，减去记录的DV前囟坐标（-2.4来自前囟）2.4毫米。 </li><li>最后，对于内侧横向（ML）面，添加2.6毫米于所记录的ML囟坐标（+2.6从囟）。对于所有的坐标一定要记录左，右坐标，因为这是一个双侧注射。 </li></ol></li><li>使针到所需的坐标。标记出针将要被插入并用钻头，钻标记区域的地方。 </li><li>用棉签无线PE走任何多余的血液或骨头碎片。 </li><li>使针的头骨，其中针的尖端接触大脑表面。然后放下针到所需的坐标慢慢地降低他们。 </li><li>一旦针是在所希望的坐标，慢慢地在5分钟（总共0.5微升）的过程中扭曲针0.1微升每分钟的柱塞注入注射器的内容物。 </li><li>一旦注入已经取得了离开再过5分钟皮层注射器。 </li></ol></li><li> 收盘涨/护理 <ol><li>取下鼠标皮质针稳定，慢慢地。然后从立体框架删除鼠标。 </li><li>应用氰基丙烯酸酯（皮肤粘合剂），以从切口皮肤的基部翼片，然后用钳子抓住皮肤的护翼，并放置在一起。 </li><li>允许氰基丙烯酸酯干燥。放在笼子里的鼠标移到一个加热垫（电热垫是，如果外科可选iCal的套装保持RT下37°C - 否则没有必要）。请务必将鼠标放在纸巾，以确保床上用品不符合手术部位。 </li><li>取决于外科手术过程的长度，确保该小鼠是从30内麻醉 - 程序在60分钟后。手术后，将动物放置在其自己笼和监控，直到它变成返回到其室收回之前意识。没有止痛剂被用于手术后的，因为它们可通过修改一些大脑生化途径的改变我们的实验的结果。然而，动物被监控，直到它们从麻醉中恢复上手术当天，和感染和疼痛/窘迫评价体征每天后操作。 </li></ol></li></ol> 3.甩头响应试验<ol><li> 建立 <ol><li>进行所有行为测试上午10:00和下午2:00，2之间 - stereota后3天CTIC注射病毒颗粒。 </li><li>溶解（±）-1-（2,5-二甲氧基-4-碘苯基）-2-氨基丙烷盐酸盐（DOI）到0.9％生理盐水溶液，以2.0毫克/公斤。还制备0.9％盐溶液中。 </li><li>制备家笼（28×18×15厘米），没有任何寝具并使用三脚架，调整摄像机，使得摄像机的视图是直接上方的家笼中。 </li><li> Habituate小鼠的空间之前在实验的开始，至少4小时。 </li><li>设置一个摄像机来记录头抽搐。 </li></ol></li><li> 实验 <ol><li>所以，这是直接在一个笼子相机位置。校准相机，使整个家庭笼中的视场。 </li><li>称重小鼠并用任一0.9％盐水或DOI的适当剂量（0.01毫升/克）腹膜内注入小鼠。注:如果鼠标的重量为25克，给予剂量为0.25毫升总体积。 </li><li>将每个鼠标背部在他们的家笼FOR 10分钟。 10分钟后，鼠标的地方入空笼的中心和验证有在视摄像头领域无盲点。按摄像机记录。离开这个房间。 注意:鼠标移动和各种行为反应其中（。甩头，耳朵划伤等请参考补充数据表1 2007年冈萨雷斯Maeso 等为通过DOI诱导行为反应的完整列表）3，将被记录30分钟。因此，存在在视记录领域无盲点是重要的。 </li><li> 30分钟后，停在原来的家笼记录在摄像机和地点鼠标背部。重复此过程对于每个鼠标。 </li></ol></li><li> 评论 <ol><li>让每个裁判审查磁带盲目鼠标的实验条件（ 即 ，头部过程中使用的药物实验抽搐或颅内注射过程中使用的病毒））。手工记录每一个甩头通过量豪特的视频。 注:头肌的定义是由鼠标（补充视频）所进行的快速摇动头部运动。 </li><li>对于每一个鼠标，平均从盲目裁判的三个总数最终HTR响应。然后基和这些值由实验条件进行统计分析（ 即 ，t检验或方差分析）。 </li></ol></li></ol>

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The authors have nothing to disclose.

连同mGlu2-KO小鼠5以前的研究结果，与mGlu2和mGlu2 / mGlu3嵌合构建的结果不形成在培养的细胞对5-HT 2A -mGlu2受体复合表明对5-HT 2A -mGlu2在异聚受体复合需要鼠标额叶皮层由LSD样致幻5-HT 2A受体激动剂诱导头部抽动行为。这种方法的一个限制是，它不会在天然组织亚细胞水平测量接近分子的接近。此外，也有应注意各种临界点。因为将小鼠用HSV病毒载体注射，即要执行的实验的时间范围是2 - 注射后4天。病毒载体的位置和表达应与初次注射后不超过4天切片脑的免疫荧光染色进行验证。小鼠中的坐标不匹配或不表达病毒载体应该可以从实验数据中排除，因为它们不表达mGlu2或mGlu2ΔTM4N。关心立体定向手术后也至关重要，因为磁头伤口不当闭合可导致感染可引起在体内实验和免疫荧光染色这两个问题。注射病毒颗粒后4 -最后，立体定向注射任何行为范式（开放场交替t检验， 等 ）后，可以，只要它是2内使用。此外，协调和表达应该通过免疫荧光确认。 该GPCR的功能均聚物和/或在活细胞中异聚现已很好地建立16-18的概念。然而，尽管取得了一些进展，还需要更多的研究来确定GPCR异聚复合物的整体动物模型19的确切作用（S）。方法，如BRET和/或FRET成像研究小动物的深部组织中的蛋白质 - 蛋白质物理接近模型可以提供手段，加强活体20 GPCR异聚的功能作用的认识。使用GPCR的HSV介导的表达任一天然的或改良的跨膜蛋白，提供在体内模型来评估的GPCR的功能和相互作用。 还需要在啮齿动物模型中进一步研究，以检查它们的部件24，以及受体内化25,26后潜在的G蛋白偶联之间21-23，结构重排的GPCR异聚的稳定性和生命时间（形成和离解）。鉴于细胞信号传导和功能的GPCR的基础性作用，它很可能是这方面的可能导致有趣的基础和翻译研究。 虽然进一步调查，需要与先前的STU定量表征在人类和小鼠的CNS两种受体的超微结构共定位，一起模具是令人信服地证明了电，蜂窝，和在小鼠模型中诱导的致幻行为反应是固有的5-HT 2A受体表达皮层锥体神经元3,27,28，这里描述利用HSV介导的表达方法获得的研究结果表明是需要由致幻5-HT 2A受体激动剂DOI诱导头部抽搐精神病样行为在小鼠额叶皮层5-HT 2A和mGlu2受体之间的异形成。 HSV介导的mGlu2的表达，但不mGlu2ΔTM4N，在mGlu2-KO小鼠的额叶皮层神经元救援由致幻5-HT 2A受体激动剂DOI感应头肌行为。这个翻译工具可能是有利的临床前研究，以评估GPCR异聚的行为表型。

致幻剂，如LSD，裸盖菇碱和麦司卡林引起人的意识，认知和情感7-9显著的变化。无论是由遗传或药物的方法羟色胺5-HT 2A受体信号的失活会导致明显减弱，以致幻剂的行为反应在两种啮齿类动物模型3,10和人类11。虽然致幻剂结合其他受体亚型8，对5-HT 2A受体被认为是必要的这些化学物质的独特的行为活动。 II组代谢型谷氨酸受体（ 即 ，mGlu2和mGlu3）一直是相当大的注意关于致幻剂的分子机制和其底层的精神病12整体作用的目标。以前，已经显示，与mGlu2蛋白（mGlu2-KO小鼠）中不表达小鼠是不敏感的HAL的细胞和行为效应lucinogens 5。还已经提出，对5-HT 2A和mGlu2受体形成一个特定的异聚复合物，通过该血清素和谷氨酸的配体调节在活细胞中的1,2- G蛋白偶联的图案。 在结构上，跨膜结构域4和mGlu2的5异的形成中发挥基础性作用与5-HT 2A受体5。此外，进一步的研究表明，三个残基位于mGlu2的TM4的细胞内端是必要的，以形成在活细胞6对5-HT 2A -mGlu2受体heterocomplex。 基于这些发现在异源表达系统中观察到，在这里，我们描述了使用野生型mGlu2和mGlu2-KO小鼠的额皮质mGlu2 / mGlu3嵌合构建的HSV介导的表达来测试是否5-HT 2A之间异形成和mGlu2是必要的通过致幻5-HT 2A受体激动剂诱导头肌的行为。

<table><tbody><tr><td>mGlu2 bicitronic herpes simplex virus (HSV) vector&amp;nbsp;</td><td>MIT Core</td><td></td><td>mGlu2 and mGlu2DTM4N were subcloned into the bicistronic HSV-GFP virus vector p1005+ HSV expressing GFP under the control of the CMV promoter. Viral particles were produced by the Viral Core Facility at the McGovern Institute (MIT). For more information, please contact the director, Dr. Rachael Neve (rneve@mit.edu)</td></tr><tr><td>mGlu2&amp;Delta;TM4N bicitronic herpes simplex virus (HSV) vector&amp;nbsp;</td><td>MIT Core</td><td></td><td>mGlu2 and mGlu2DTM4N were subcloned into the bicistronic HSV-GFP virus vector p1005+ HSV expressing GFP under the control of the CMV promoter. Viral particles were produced by the Viral Core Facility at the McGovern Institute (MIT). For more information, please contact the director, Dr. Rachael Neve (rneve@mit.edu)</td></tr><tr><td>GFP bicitronic herpes simplex virus (HSV) vector&amp;nbsp;</td><td>MIT Core</td><td></td><td>mGlu2 and mGlu2DTM4N were subcloned into the bicistronic HSV-GFP virus vector p1005+ HSV expressing GFP under the control of the CMV promoter. Viral particles were produced by the Viral Core Facility at the McGovern Institute (MIT). For more information, please contact the director, Dr. Rachael Neve (rneve@mit.edu)</td></tr><tr><td>Xylazine&amp;nbsp;</td><td>Lloyd</td><td>List no. 4811-20ml, NADA #139-236, NDC Code(s): 61311-481-10</td><td>1.35 ml of ketamine (100 mg/ml) + 0.75 ml of xylazine (20 mg/ml) are diluted in 12.0 ml of 0.9% saline solution</td></tr><tr><td>Ketamine&amp;nbsp;</td><td>Vedco</td><td>KetaVed-10ml, NADA #200-029, NDC Code(s): 50989-161-06</td><td>1.35 ml of ketamine (100 mg/ml) + 0.75 ml of xylazine (20 mg/ml) are diluted in 12.0 ml of 0.9% saline solution</td></tr><tr><td>Ophthalmic gel</td><td>Fisher Scientific</td><td>NC0550805</td><td></td></tr><tr><td>Burret clips</td><td>Fisher Scientific</td><td>NC9268369</td><td></td></tr><tr><td>Feather surgical blade</td><td>Fisher Scientific</td><td>NC9032736</td><td></td></tr><tr><td>Hydrogen Peroxide</td><td>Fisher Scientific</td><td>19-898-919&amp;nbsp;</td><td></td></tr><tr><td>Hamilton syringe</td><td>Fisher Scientific</td><td>14815203</td><td></td></tr><tr><td>Hamilton&amp;trade; Small Hub Removable Needles (33 Ga)</td><td>Fisher Scientific</td><td>14816206</td><td></td></tr><tr><td>Cordless Micro Drill</td><td>Fisher Scientific</td><td>NC9089241</td><td></td></tr><tr><td>Dermabond Dermal Adhesive</td><td>Fisher Scientific</td><td>NC0690470</td><td></td></tr><tr><td>(&amp;plusmn;)-1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI)</td><td>Sigma-Aldrich</td><td>42203-78-1</td><td>Dissolved in 0.9% saline solution to the concentration of 2.0 mg/kg</td></tr></tbody></table>

hsv-mediated transgene expression of chimeric constructs to study behavioral function of gpcr heteromers in mice

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2. Consequently, investigation of structural details of the interaction between 5-HT2A and mGlu2 affecting schizophrenia-related behaviors represents a powerful translational tool. As previously shown, the head-twitch response (HTR) in mice is elicited by hallucinogenic drugs and this behavioral response is absent in 5-HT2A knockout (KO) mice3,4. Additionally, by conditionally expressing the 5-HT2A receptor only in cortex, it was demonstrated that 5-HT2A receptor-dependent signaling pathways on cortical pyramidal neurons are sufficient to elicit head-twitch behavior in response to hallucinogenic drugs3. Finally, it has been shown that the head-twitch behavioral response induced by the hallucinogens DOI and lysergic acid diethylamide (LSD) is significantly decreased in mGlu2-KO mice5. These findings suggest that mGlu2 is at least in part necessary for the 5-HT2A receptor-dependent psychosis-like behavioral effects induced by LSD-like drugs. However, this does not provide evidence as to whether the 5-HT2A-mGlu2 receptor complex is necessary for this behavioral phenotype. To address this question, herpes simplex virus (HSV) constructs to express either mGlu2 or mGlu2&#916;TM4N (mGlu2/mGlu3 chimeric construct that does not form the 5-HT2A-mGlu2 receptor complex) in the frontal cortex of mGlu2-KO mice were used to examine whether this GPCR heteromeric complex is needed for the behavioral effects induced by LSD-like drugs6.

以前的研究结果表明，该头部抽搐鼠行为反应被可靠和鲁棒由致幻剂引起，并且它在5-HT 2A -KO小鼠3是不存在。此外，已经表明由致幻5-HT 2A激动剂DOI和LSD引起头部抽搐反应在mGlu2-KO小鼠5被显著降低。然而，虽然以前的研究结果令人信服地证明，5-HT 2A和mGlu2被组装成在转染细胞1,2,15 体外异聚复合物，此结构布置是否表现为这样在活小鼠中仍未解决。要充分认识在mGlu2-KO小鼠额叶皮质致幻5-HT 2A受体激动剂，无论是mGlu2或mGlu2ΔTM4N表达引起的精神样作用的5-HT 2A受体-mGlu2的heterocomplex的作用，研究这是否MANIPulation调节行为。 小鼠接受双顺反子的HSV帧内额叶皮质注射表达单独绿色荧光蛋白（GFP），要么mGlu2或mGlu2ΔTM4N，或绿色荧光蛋白。首先，它证实了病毒过表达在小鼠额叶皮质mGlu2或mGlu2ΔTM4N（ 图1A和1B）。正如以前证明5，通过DOI诱导头部抽搐的行为与空载体HSV-GFP注射mGlu2-KO小鼠中不存在。值得注意的是，由致幻5-HT 2A激动剂DOI诱导头部抽搐反应在mGlu2-KO小鼠过表达mGlu2获救，但不mGlu2ΔTM4N，额叶皮层相比，在表达GFP（ 图1C）的动物看到。总之，这些研究结果表明，在额皮质中的5-HT 2A -mGlu2受体复合物是用于调节精神病样状态的关键。 图1. mGlu2表达的 mGlu2 的受体Heterocomplex。表达与5-HT 2A受体是必要的致幻通过诱导药物精神病样的行为。 （ 一 ）额叶皮层HSV介导的转基因表达的形象代表。 HSV-mGlu2，这也表达绿色荧光蛋白，注入额叶皮层，和GFP表达，免疫细胞化学，比例尺，200微米透露。 （B）中的HSV介导的转基因表达在mGlu2-KO小鼠的小鼠额叶皮层和抗mGlu2反应性通过Western印迹测定。对mGlu2受体第一抗体的特异性先前已证实了在敲除小鼠6。代谢型谷氨酸受体是形成共价连接的同二聚体的GPCR。我们测量mGlu2的免疫反应性作为单体（100 kDa）的6。 （C </stronmGlu2克&gt;）病毒介导的表达，但不mGlu2ΔTM4N，在mGlu2- KO小鼠的额皮质显著抢救由致幻5-HT 2A激动剂DOI（每组n = 4）诱导的头部抽搐反应。 *** P &lt;0.001; NS，不显著;邦费罗尼的的单向ANOVA事后检验。误差棒代表SEM图由莫雷诺等人 （2012）修改6。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53717/53717fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53717/HeadTwitchResponse.wmv" target="_blank">补充视频1.甩头响应。 （点击下载）。</a> CD-1野生型小鼠用2.0毫克/千克DOI注射，并置于笼（壁涂黑两个笼之间）以引发头颤搐反应（后行为引起*）。

Watch this Scientific Journal Video about HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice at JoVE.com

HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice

本文介绍如何注入病毒载体插入鼠标额叶皮质测试需要GPCR异形成行为检测。

研究小鼠GPCR异聚的行为功能嵌合构造的HSV介导的转基因表达

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2.  ...

hsv-mediated-transgene-expression-chimeric-constructs-to-study

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

7.2K 次观看.  Icahn School of Medicine at Mount Sinai. 本文介绍如何注入病毒载体插入鼠标额叶皮质测试需要GPCR异形成行为检测。 

视频: HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration of transgenes. However, due to the low efficiency of homologous recombination (HR) and various indel mutations of non-homologous end joining (NHEJ)-based strategies in non-dividing cells, in vivo genome editing remains a great challenge. Here, we describe a homology-mediated end joining (HMEJ)-based CRISPR/Cas9 system for efficient in vivo precise targeted integration. In this system, the targeted genome and the donor vector containing homology arms (~800 bp) flanked by single guide RNA (sgRNA) target sequences are cleaved by CRISPR/Cas9. This HMEJ-based strategy achieves efficient transgene integration in mouse zygotes, as well as in hepatocytes in vivo. Moreover, a HMEJ-based strategy offers an efficient approach for correction of fumarylacetoacetate hydrolase (Fah) mutation in the hepatocytes and rescues Fah-deficiency induced liver failure mice. Taken together, focusing on targeted integration, this HMEJ-based strategy provides a promising tool for a variety of applications, including generation of genetically modified animal models and targeted gene therapies.

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system provides a promising tool for genetic engineering, and opens up the possibility of targeted integration of transgenes. We describe a homology-mediated end joining (HMEJ)-based strategy for efficient DNA targeted integration in vivo and targeted gene therapies using CRISPR/Cas9.

CRISPR/Cas9-mediated 目标集成在体内使用基于同源介导的端接连接策略

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration ...

科研

遗传学

Lentiviral Mediated Production of Transgenic Mice: A Simple and Highly Efficient Method for Direct Study of Founders

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a routine procedure is widely utilized throughout the world and its main limitation resides in the poor efficacy of transgene integration, resulting in a low yield of founder animals. Only few percent of animals born after implantation of injected fertilized oocytes have integrated the transgene. In contrast, lentiviral vectors are powerful tools for integrative gene transfer and their use to transduce fertilized oocytes allows highly efficient production of founder transgenic mice with an average yield above 70%. Furthermore, any mouse strain can be used to produce transgenic animal and the penetrance of transgene expression is extremely high, above 80% with lentiviral mediated transgenesis compared to DNA microinjection. The size of the DNA fragment that can be cargo by the lentiviral vector is restricted to 10 kb and represents the major limitation of this method. Using a simple and easy to perform injection procedure beneath the zona pellucida of fertilized oocytes, more than 50 founder animals can be produced in a single session of microinjection. Such a method is highly adapted to perform, directly in founder animals, rapid gain and loss of function studies or to screen genomic DNA regions for their ability to control and regulate gene expression in vivo.

Here, we present a protocol to promote transgene integration and production of founder transgenic mice with high efficacy by a simple injection of a lentiviral vector in the perivitelline space of a fertilized oocyte.

慢病毒载体介导的转基因小鼠生产: 一种简单高效的创始人直接研究方法

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a ...

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the olfactory bulb (OB). The molecular mechanisms of OSN axon growth and targeting are not well understood. Manipulating gene expression and subsequent 
visualizing of single OSN axons and their terminal arbor morphology have thus far been challenging. To study gene function at the single cell level within a specified 
time frame, we developed a lentiviral based technique to manipulate gene expression in OSNs in vivo. Lentiviral particles are delivered to OSNs by microinjection 
into the olfactory epithelium (OE). Expression cassettes are then permanently integrated into the genome of transduced OSNs. Green fluorescent protein expression 
identifies infected OSNs and outlines their entire morphology, including the axon terminal arbor. Due to the short turnaround time between microinjection and 
reporter detection, gene function studies can be focused within a very narrow period of development. With this method, we have detected GFP expression within as 
few as three days and as long as three months following injection. We have achieved both over-expression and shRNA mediated knock-down by lentiviral microinjection. 
This method provides detailed morphologies of OSN cell bodies and axons at the single cell level in vivo, and thus allows characterization of candidate gene function 
during olfactory development.

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

We present a lentiviral technique for genetic manipulation and visualization of single olfactory sensory neuron axon and its terminal arborization in vivo.

嗅觉感官神经元的慢病毒介导的遗传操作和可视化在体内</em

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the ...

神经科学

Recurrent Herpetic Stromal Keratitis in Mice, a Model for Studying Human HSK

Herpetic eye disease, termed herpetic stromal keratitis (HSK), is a potentially blinding infection of the cornea that results in over 300,000 clinical visits each year for treatment. Between 1 and 2 percent of those patients with clinical disease will experience loss of vision of the infected cornea. The vast majority of these cases are the result of reactivation of a latent infection by herpes simplex type I virus and not due to acute disease. Interestingly, the acute infection is the model most often used to study this disease. However, it was felt that a recurrent model of HSK would be more reflective of what occurs during clinical disease. The recurrent animal models for HSK have employed both rabbits and mice. The advantage of rabbits is that they experience reactivation from latency absent any known stimulus. That said, it is difficult to explore the role that many immunological factors play in recurrent HSK because the rabbit model does not have the immunological and genetic resources that the mouse has. We chose to use the mouse model for recurrent HSK because it has the advantage of there being many resources available and also we know when reactivation will occur because reactivation is induced by exposure to UV-B light. Thus far, this model has allowed those laboratories using it to define several immunological factors that are important to this disease. It has also allowed us to test both therapeutic and vaccine efficacy.

Most studies of herpetic corneal disease use a primary infection model. However, primary infection with HSV-1 does not typically lead to human disease. Here we describe a recurrent model of herpetic corneal disease, which more closely mimics human disease.

复发性单纯疱疹性角膜基质性角膜炎小鼠模型研究人类HSK

Herpetic eye disease, termed herpetic stromal keratitis (HSK), is a potentially blinding infection of the cornea that results in over 300,000 clinical ...

免疫与感染

Viral Tracing of Genetically Defined Neural Circuitry

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus (RABV), and more recently vesicular stomatitis virus (VSV), for studying circuitry, is becoming increasingly popular. These higher throughput methods use viruses that transmit between neurons in either the anterograde or retrograde direction.
Recently, a modified RABV for monosynaptic retrograde tracing was developed. (Figure 1A). In this method, the glycoprotein (G) gene is deleted from the viral genome, and resupplied only in targeted neurons. Infection specificity is achieved by substituting a chimeric G, composed of the extracellular domain of the ASLV-A glycoprotein and the cytoplasmic domain of the RABV-G (A/RG), for the normal RABV-G1. This chimeric G specifically infects cells expressing the TVA receptor1. The gene encoding TVA can been delivered by various methods2-8. Following RABV-G infection of a TVA-expressing neuron, the RABV can transmit to other, synaptically connected neurons in a retrograde direction by nature of its own G which was co-delivered with the TVA receptor. This technique labels a relatively large number of inputs (5-10%)2 onto a defined cell type, providing a sampling of all of the inputs onto a defined starter cell type.
We recently modified this technique to use VSV as a transsynaptic tracer9. VSV has several advantages, including the rapidity of gene expression. Here we detail a new viral tracing system using VSV useful for probing microcircuitry with increased resolution. While the original published strategies by Wickersham et al.4 and Beier et al.9 permit labeling of any neurons that project onto initially-infected TVA-expressing-cells, here VSV was engineered to transmit only to TVA-expressing cells (Figure 1B). The virus is first pseudotyped with RABV-G to permit infection of neurons downstream of TVA-expressing neurons. After infecting this first population of cells, the virus released can only infect TVA-expressing cells. Because the transsynaptic viral spread is limited to TVA-expressing cells, presence of absence of connectivity from defined cell types can be explored with high resolution. An experimental flow chart of these experiments is shown in Figure 2. Here we show a model circuit, that of direction-selectivity in the mouse retina. We examine the connectivity of starburst amacrine cells (SACs) to retinal ganglion cells (RGCs).

A method of tracing synaptically connected neurons is described. We use TVA specificity of an upstream cell to probe whether a cell population of interest receives synaptic input from genetically defined cell types.

病毒遗传背景明确的神经回路跟踪

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus ...

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

立体定向注射病毒载体的基因操作在小鼠脊髓条件

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

Somatic Genome-Engineered Mouse Models Using In Vivo Microinjection and Electroporation

Germline genetically engineered mouse models (G-GEMMs) have provided valuable insight into in vivo gene function in development, homeostasis, and disease. However, the time and cost associated with colony creation and maintenance are high. Recent advances in CRISPR-mediated genome editing have allowed the generation of somatic GEMMs (S-GEMMs) by directly targeting the cell/tissue/organ of interest.
The oviduct, or fallopian tube in humans, is considered the tissue-of-origin of the most common ovarian cancer, high-grade serous ovarian carcinomas (HGSCs). HGSCs initiate in the region of the fallopian tube distal to the uterus, located adjacent to the ovary, but not the proximal fallopian tube. However, traditional mouse models of HGSC target the entire oviduct, and thus do not recapitulate the human condition. We present a method of DNA, RNA, or ribonucleoprotein (RNP) solution microinjection into the oviduct lumen and in vivo electroporation to target mucosal epithelial cells in restricted regions along the oviduct. There are several advantages of this method for cancer modeling, such as 1) high adaptability in targeting the area/tissue/organ and region of electroporation, 2) high flexibility in targeted cell types (cellular pliancy) when used in combination with specific promoters for Cas9 expression, 3) high flexibility in the number of electroporated cells (relatively low frequency), 4) no specific mouse line is required (immunocompetent disease modeling), 5) high flexibility in gene mutation combination, and 6) possibility of tracking electroporated cells when used in combination with a Cre reporter line. Thus, this cost-effective method recapitulates human cancer initiation.

Somatic Genome-Engineered Mouse Models Using In Vivo Microinjection and Electroporation

This protocol describes microinjection and in vivo electroporation for regionally restricted CRISPR-mediated genome editing in the mouse oviduct.

使用 体内 显微注射和电穿孔的体细胞基因组工程小鼠模型

Germline genetically engineered mouse models (G-GEMMs) have provided valuable insight into in vivo gene function in development, homeostasis, and disease. ...

癌症研究

A Custom Multiphoton Microscopy Platform for Live Imaging of Mouse Cornea and Conjunctiva

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. Multiphoton microscopy (MPM) has become one of the most popular imaging modalities for biomedical study at cellular levels in vivo, advantages include high resolution, deep tissue penetration and minimal phototoxicity. We have designed an MPM imaging platform with a customized mouse eye holder and a stereotaxic stage for imaging ocular surface in vivo. Dual fluorescent protein reporter mouse enables visualization of cell nuclei, cell membranes, nerve fibers, and capillaries within the ocular surface. In addition to multiphoton fluorescence signals, acquiring second harmonic generation (SHG) simultaneously allows for the characterization of collagenous stromal architecture. This platform can be employed for intravital imaging with accurate positioning across the entire ocular surface, including cornea and conjunctiva.

Presented here is a multiphoton microscopic platform for live mouse ocular surface imaging. Fluorescent transgenic mouse enables the visualization of cell nuclei, cell membranes, nerve fibers and capillaries within the ocular surface. Non-linear second harmonic generation signals derived from collagenous structures provide label-free imaging for stromal architectures.

用于小鼠角膜和康康菲瓦实时成像的自定义多光子显微镜平台

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. ...

生物工程

Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or triple transgenic mice are generated in order to achieve selective expression of a reporter or effector gene (e.g., from the Rosa26 locus) in the desired spinal population. 2) Intraspinal injection of Cre-dependent recombinant adeno-associated virus (rAAV); here Cre-dependent AAV vectors coding for the reporter or effector gene of choice are injected into the spinal cord of mice expressing Cre recombinase in the desired neuronal subpopulation. This protocol describes how to generate Cre-dependent rAAV vectors and how to transduce neurons in the dorsal horn of the lumbar spinal cord segments L3-L5 with rAAVs. As the lumbar spinal segments L3-L5 are innervated by those peripheral sensory neurons that transmit sensory information from the hindlimbs, spontaneous behavior and responses to sensory tests applied to the hindlimb ipsilateral to the injection side can be analyzed in order to interrogate the function of the manipulated neurons in sensory processing. We provide examples of how this technique can be used to analyze genetically defined subsets of spinal neurons. The main advantages of virus-mediated transgene expression in Cre transgenic mice compared to classical reporter mouse-induced transgene expression are the following: 1) Different Cre-dependent rAAVs encoding various reporter or effector proteins can be injected into a single Cre transgenic line, thus overcoming the need to create several multiple transgenic mouse lines. 2) Intraspinal injection limits manipulation of Cre-expressing cells to the injection site and to the time after injection. The main disadvantages are: 1) Reporter gene expression from rAAVs is more variable. 2) Surgery is required to transduce the spinal neurons of interest. Which of the two methods is more appropriate depends on the neuron population and research question to be addressed.

Intraspinal injection of recombinase dependent recombinant adeno-associated virus (rAAV) can be used to manipulate any genetically labelled cell type in the spinal cord. Here we describe how to transduce neurons in the dorsal horn of the lumbar spinal cord. This technique enables functional interrogation of the manipulated neuron subtype.

腺相关病毒介导的脊髓基因神经元的转基因表达

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or ...

<meta charset="utf8"></head><body>向导 RNA 的逆转录病毒转导用于 Th17 分化中的基因编辑

Retroviral CRISPR/Cas9-Mediated Gene Targeting for the Study of Th17 Differentiation in Vitro

T helper cells that produce IL-17A, known as Th17 cells, play a critical role in immune defense and are implicated in autoimmune disorders. CD4 T cells can be stimulated with antigens and well-defined cytokine cocktails in vitro to mimic Th17 cell differentiation in vivo. Research has been conducted extensively on the Th17 differentiation regulation mechanisms using the in vitro Th17 polarization assay.
Conventional Th17 polarization methods typically involve obtaining na&#239;ve CD4 T cells from genetically modified mice to study the effects of specific genes on Th17 differentiation and function. These methods can be time-consuming and costly and may be influenced by cell-extrinsic factors from the knockout animals. Thus, a protocol using retroviral transduction of guide RNA to introduce gene knockout in CRISPR/Cas9 knockin primary mouse T cells serves as a very useful alternative approach. This paper presents a protocol to differentiate na&#239;ve primary T cells into Th17 cells following retroviral-mediated gene targeting, as well as the subsequent flow cytometry analysis methods for assaying infection and differentiation efficiency.

<meta charset="utf8"></head><body>逆转录病毒 CRISPR/Cas9 介导的基因靶向在体外研究 Th17 分化

We present a protocol for retroviral transduction of guide RNA into primary T cells from Cas9 transgenic mice, providing an efficient alternative for gene editing in studying Th17 differentiation.

逆转录病毒 CRISPR/Cas9 介导的基因靶向在体外研究 Th17 分化

T helper cells that produce IL-17A, known as Th17 cells, play a critical role in immune defense and are implicated in autoimmune disorders. CD4 T cells ...

研究小鼠GPCR异聚的行为功能嵌合构造的HSV介导的转基因表达

摘要

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此视频中的章节

CRISPR/Cas9-mediated 目标集成在体内使用基于同源介导的端接连接策略

慢病毒载体介导的转基因小鼠生产: 一种简单高效的创始人直接研究方法

嗅觉感官神经元的慢病毒介导的遗传操作和可视化在体内

复发性单纯疱疹性角膜基质性角膜炎小鼠模型研究人类HSK

病毒遗传背景明确的神经回路跟踪

立体定向注射病毒载体的基因操作在小鼠脊髓条件

使用体内显微注射和电穿孔的体细胞基因组工程小鼠模型

用于小鼠角膜和康康菲瓦实时成像的自定义多光子显微镜平台

腺相关病毒介导的脊髓基因神经元的转基因表达

逆转录病毒 CRISPR/Cas9 介导的基因靶向在体外研究 Th17 分化

研究小鼠GPCR异聚的行为功能嵌合构造的HSV介导的转基因表达

摘要

探索更多视频

此视频中的章节

CRISPR/Cas9-mediated 目标集成在体内使用基于同源介导的端接连接策略

慢病毒载体介导的转基因小鼠生产: 一种简单高效的创始人直接研究方法

嗅觉感官神经元的慢病毒介导的遗传操作和可视化在体内

复发性单纯疱疹性角膜基质性角膜炎小鼠模型研究人类HSK

病毒遗传背景明确的神经回路跟踪

立体定向注射病毒载体的基因操作在小鼠脊髓条件

使用 体内 显微注射和电穿孔的体细胞基因组工程小鼠模型

用于小鼠角膜和康康菲瓦实时成像的自定义多光子显微镜平台

腺相关病毒介导的脊髓基因神经元的转基因表达

逆转录病毒 CRISPR/Cas9 介导的基因靶向在体外研究 Th17 分化

使用体内显微注射和电穿孔的体细胞基因组工程小鼠模型